Recombinant FGF21 as a novel approach for treating ischemic stroke in type 2 diabetes

项目摘要 糖尿病卒中患者的死亡率更高，神经功能结局更差。新兴的临床和 实验数据表明，血脑屏障破坏、神经炎症和中风恢复障碍是 在T2 D中恶化。因此，本项目的目标是研究治疗方法， 这些特定的T2 D机制。成纤维细胞生长因子-21（FGF 21）是一种循环内分泌因子， 主要在肝脏中表达的激素。FGF 21具有多效性代谢作用，但也具有组织 保护和修复作用。在这里，我们将测试外源性重组人源化的总体假设， FGF 21（rFGF 21）激活FGFR 1-β-klotho复合物，可改善T2 D影响的机制， 脑卒中通过：（1）激活PPAR γ以保护BBB，（2）抑制NF κ B介导但激活PPAR γ 对于神经炎症调节，（3）促进AMPK/Nrf 2介导的血管/白色物质重塑， 促进中风后的神经恢复。 目标1。研究rFGF 21对T2 D中风小鼠中血脑屏障完整性的影响。我们将检测FGF 21 特异性受体FGFR 1磷酸化、PPAR γ活性、BBB完整性、出血性转化 血管炎性分子和紧密连接蛋白的表达，在给药后最多3天进行评估。 在db/db T2 D小鼠中的中风（dMCAO）和脑内皮/星形胶质细胞共培养物的体外模型中的T2 D中风。 目标二。研究rFGF 21对T2 D卒中中神经炎症的影响。我们将检测NF κ B B和PPAR γ 激活，以及与小胶质细胞/巨噬细胞激活，促炎因子表达的相关性， 在局灶性dMCAO卒中后通过RT-PCR、免疫组化、western印迹和流式细胞术测定的病变面积 T2 D小鼠和体外原代小胶质细胞培养物中。目标3。研究rFGF 21对血管/白色 2型糖尿病卒中的物质重塑我们将检测AMPK和Nrf 2的激活， 分离的脑微血管片段和脑组织，通过蛋白质印迹分析，免疫组织化学， mRNA微阵列。我们还将测试血管和白色物质重塑的生物标志物， T2 D小鼠缺血性中风后的免疫组织化学、RT-PCR和蛋白质印迹，以及T2 D小鼠缺血性中风后的体外模型。 原代人脑内皮细胞培养物和少突胶质细胞培养物。

Project Summary Diabetic stroke patients have higher mortality and worse neurological outcomes. Emerging clinical and experimental data suggest that BBB disruption, neuroinflammation, and stroke recovery impairment are exacerbated in T2D. Hence, the goal of this project is to investigate therapeutic approaches that can target these specific T2D mechanisms in stroke. Fibroblast Growth Factor-21 (FGF21) is a circulating endocrine hormone that is primarily expressed in the liver. FGF21 has pleiotropic metabolic actions, but also has tissue protective and repair roles. Here, we will test the overall hypothesis that exogenous recombinant human FGF21 (rFGF21) activates FGFR1--klotho complex that may ameliorate T2D-affected mechanisms after stroke via: (1) activation of PPAR for BBB protection, (2) inhibition of NFB-mediated but activation of PPAR for neuroinflammation modulation, (3) promotion of AMPK/Nrf2-mediated vascular/white matter remodeling to boost neurorestoration after stroke. Aim 1. Investigate rFGF21 effects on blood-brain barrier integrity in T2D stroke mice. We will examine FGF21 specific receptor FGFR1 phosphorylation, PPAR activity, BBB integrity, hemorrhagic transformation, expression of vascular inflammatory molecules and tight junction proteins, are assessed up to 3 days after stroke (dMCAO) in db/db T2D mice, and T2D stroke in vitro model of brain endothelial/astrocytes co-cultures. Aim 2. Investigate rFGF21 effects on neuroinflammation in T2D stroke. We will examine NFB and PPAR activation, and correlation with microglia/macrophage activation, pro-inflammatory factor expression in peri- lesion area by RT-PCR, immunohistochemistry, western blots, and flow cytometry after focal dMCAO stroke in T2D mice, and in primary microglia cultures in vitro. Aim 3. Investigate rFGF21 effects on vascular/white matter remodeling in T2D stroke. We will examine activation of AMPK and Nrf2, expression of trophic factors in isolated brain microvascular fragments and brain tissues by western blot analysis, immunohistochemistry, and mRNA microarray. We will also test biomarkers of vascular and white matter remodeling over time by immunohistochemistry, RT-PCR and western blots after ischemic stroke of T2D mice, and in vitro models of primary human brain endothelial cultures and oligodendrocyte cultures.